The long term goal of this work is primarily to understand the molecular mechanisms that produce mutations when mutagen-damaged DNA is replicated in E. coli, and secondarily to investigate other processes that promote tolerance of DNA damage. Although induced mutagenesis, and damage tolerance in general, is regulated very differently in E. coli and eukaryotes, basic enzymological processess are likely to be similar. Information of significance to human genetic diseases, such as cancer, can therefore be gained from studies employing the unrivalled experimental tools available with the bacterium. The aim of this project is to analyze the processes in vivo, by employing the analytical power of experiments in which vectors carrying defined and specifically located lesions are transfected into some of the many well characterized mutant strains that are available with the bacterium. Specific aims include: indentification of the gene products that are responsible for efficient replication past a T-T dimer in cells lacking DNA polymerase II, IV, and V and proofreading activity; identification of the MucB residues responsible for its differences in mutagenic properties comared to pol V; investigation of the RecA-independent recombination mechanism for the error free bypass of the T_T pyrimidine (6-4) pyrimidinone UV-photoproduct; measurement of the deamination rates in vivo of cytosine within a TC cis-syn cyclobutane dimer; and determination of the structure of duplex oligonucleotide molecules that contain a T_C pyrimidine (6-4) pyrimidinone adduct and the dewar isomers of the T-T and T-C (6-4) photoproducts, using nuclear magnetic resonance. The first of these aims is concerned with identifying the DNA polymerase or accessory factor capable of promoting replication past a T-T dimer in the absence of enzymes usually associated with SOS mutagenesis, whereas the second goal is to explore the structural reasons for the markedly different properties of related polymerases. The third aim is designed to explore what appears to be a novel, RecA independent, recombination process, while the last two investigate the reasons for the high mutability and specificity of two important UV photoproducts.